PROJECT SUMMARY Cancer research and treatment are entering the era of precision medicine which demands better biomarkers and tests to improve the abilities to measure risk, diagnose disease, and predict patient prognosis and therapy response. Recently, cell-free microRNAs (miRNAs) found in the peripheral circulation of cancer patients have attracted immense interest owing to their promising potential as non-invasive biomarkers to detect cancer earlier and assist in individualizing therapeutic treatment. Despite the promise of circulating miRNAs, moving from proof-of-concept to clinical practice remains a work in progress. One roadblock is the complex, heterogeneous origin of circulating miRNAs which confounds the identification of disease-specific signatures. Here we propose to decipher the complexity of circulating miRNA profiles by targeting exosomes, a major type of miRNA carriers. Exosomes are one type of extracellular vesicles (EVs) secreted by most cells and have been implicated in many biological processes, including tumor progression, metastasis, and drug resistance. Thus probing tumor-derived exosomes provides a promising route to develop reliable miRNA biomarkers. However, exosome analysis remains a daunting challenge and there is a lack of reliable methods for analyzing tumor-derived, circulating exosomes and their cargo. Current isolation and analysis methods are expensive, lack specificity to exosome subtypes, consume large sample volumes, and cause vast variations in the downstream analysis of exosomal miRNAs (exo-miRNA). To address these technological challenges, here we propose to develop a Microfluidic Circulating MicroRNA Analysis Platform (CMAP). The CMAP presents for the first time a Lab-on-a-Chip system for integrative profiling of circulating miRNAs, which synergistically integrates the innovative exosome isolation technique and quantitative miRNA profiling technology established by the PIs? labs. The proposed goals will be achieved via the following two specific aims: 1) develop and optimize a microfluidic circulating microRNA analysis platform (CMAP); and 2) establish the CMAP for clinical development of circulating exosomal miRNAs as a non-invasive liquid biopsy method for early cancer diagnosis. Overall, these studies will immediately address the unmet urgent need of new analytical methodologies and technologies for non-invasive circulating miRNA profiling to accelerate the development of clinically viable miRNA biomarkers. The proposed studies represent a first-step towards the PIs? long-term research interest in basic and translational research to develop liquid biopsy-based markers and tests for non- invasive early diagnosis and precision treatment of cancer. While designed to meet specific need in OvCa diagnosis, the CMAP will ultimately provide a transformative and versatile platform technology for basic and clinical investigation of a wide range of malignancies.